1. Field of the Invention
The present invention is in the field of cancer therapeutics, diagnostics, and drug metabolism. In particular, the present invention relates to characterization of the genetic basis for thiopurine methyltransferase deficiency. Three separate point mutations are, at least in part, responsible for severe hematopoietic toxicity in cancer patients who are treated with standard dosages of 6-mercaptopurine, 6-thioguanine or azathioprine.
2. Related Art
Thiopurine methyltransferase (TPMT, E.C. 2.1.1.67) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including the anticancer agents 6-mercaptopurine (6MP) and 6-thioguanine, and the immunosuppressant azathioprine. TPMT activity exhibits genetic polymorphism, with approximately 89% of Caucasians and African-Americans having high TPMT activity, 11% intermediate activity (presumed heterozygotes), and approximately one in 300 inheriting TPMT-deficiency as an autosomal recessive trait. (Weinshilboum, R. M. and Sladek, S. L., Am. J. Hum. Genet. 32:651-662 (1980); McLeod, H. L. et al., Clin. Pharmacol. Ther. 55:15-20 (1994)). TPMT activity is typically measured in erythrocytes, as the level of TPMT activity in human liver, kidney, lymphocytes and leukemic lymphoblast correlates with that in erythrocytes (Van Loon, J. A. and Weinshilboum, R. M., Biochem. Genet. 20:637-658 (1982); Szumlanski, C. L., et al., Pharmacogenetics 2:148-159 (1992); McLeod, H. L. et al., Blood 85:1897-1902 (1995)).
Mercaptopurine, thioguanine, and azathioprine are prodrugs with no intrinsic activity, requiring intracellular conversion to thioguanine nucleotides (TGN), with subsequent incorporation into DNA, as one mechanism of their antiproliferative effect (Lennard, L., Eur. J. Clin. Pharmacol 43:329-339 (1992)). Alternatively, these drugs are metabolized to 6-methyl-mercaptopurine (MeMP) or 6-methyl-thioguanine (MeTG) by TPMT or to 6-thiouric acid (6TU) by xanthine oxidase; MeMP, MeTG, and 6TU are inactive metabolites. Thus, metabolism of 6MP, azathioprine, or thioguanine by TPMT shunts drug away from the TGN activation pathway. Clinical studies with 6MP and azathioprine have established an inverse correlation between erythrocyte TPMT activity and erythrocyte TGN accumulation, indicating that patients who less efficiently methylate these thiopurines have more extensive conversion to thioguanine nucleotides (Lennard, L., et al., Lancet 336:225-229 (1990); Lennard, L. et al., Clin. Pharmacol. Ther. 46:149-154 (1989)). Moreover, patients with TPMT deficiency accumulate significantly higher erythrocyte TGN if treated with standard dosages of 6MP or azathioprine, leading to severe hematopoietic toxicity, unless the thiopurine dosage is lowered substantially (e.g. 8-15 fold reduction) (Evans, W. E., et al., J. Pediatr. 19:985-989 (1991); McLeod, H. L., et al., Lancet 341:1151 (1993); Lennard, L., et al., Arch. Dis. Child. 69:577-579 (1993)). The majority of such patients are identified only after experiencing severe toxicity, even though prospective measurement of erythrocyte TPMT activity has been advocated by some (Lennard, L. et al., Clin. Pharmacol. Ther. 41:18-25 (1987)). Unfortunately, TPMT assays are not widely available and newly diagnosed patients with leukemia or organ transplant recipients are frequently given erythrocyte transfusions, precluding measurement of their constitutive TPMT activity before thiopurine therapy is initiated. Alternatively, if the inactivating mutations of the human TPMT gene can be identified, PCR-based methods can be developed to determine TPMT genotype and prospectively predict phenotype, as is now possible for drug metabolizing enzymes such as debrisoquin-hydroxylase (Heim, M. and Meyer, U. A., Lancet 336:529-532 (1990)) and N-acetyltransferase (Grant, D. M., Pharmacogenetics 3:45-50 (1993)).
Identification of the predominant mutations at the TPMT locus would not only offer a strategy for prospectively identifying heterozygotes and TPMT-deficient patients based on their genotype, prior to treatment with potentially toxic dosages of mercaptopurine, azathioprine and thioguanine, it would also provide important insights into the molecular mechanisms of this genetic polymorphism.